The cDNA encoding a member of a family of steroid UDP-glucuronosyltransferases has been cloned and sequenced. The 1871-base pair (bp) cDNA, designated UDPGTr-5, contains an open reading frame of 1590 bp flanked by 43 and 238 bp of 5'- and 3'-untranslated regions, respectively. The 530-amino acid protein encoded by the open reading frame is 93% and 84% similar in sequence to UDPGTr-3 and UDPGTr-4, which are other members of this multigene family. In common with these forms, the encoded protein contains regions characteristic of a signal peptide and a transmembrane-anchoring domain at the carboxyl terminus. Potential asparagine-linked glycosylation sites are not present in the encoded protein. Expression of UDPGTr-5 cDNA in COS cells demonstrated that the encoded enzyme has a subunit size of 50 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The expressed enzyme glucuronidates testosterone and dihydrotestosterone, although its activity towards these two substrates is about 30-fold less than that of UDPGTr-3. This low activity appeared to be a bone fide property of the enzyme and was not due to lower amounts of protein synthesized or increased rates of protein degradation. Northern analysis and cDNA sequencing demonstrated that UDPGTr-5 is encoded by two mRNA species which differed in the lengths of their 3'-untranslated regions. The cDNA to the longer transcript has a 1768-bp 3'-untranslated region which contains a segment that is 80% similar to the rat identifier sequence (ID) family of repetitive DNA elements. These data indicate that UDPGTr-5 is another 17 beta-hydroxysteroid UDP-glucuronosyltransferase that is encoded by two mRNAs transcribed from a single gene.